Enhanced antibody production in mice to the malaria antigen AMA1 by CPG 7909 requires physical association of CpG and antigen

Abstract

CpG oligodeoxynucleotides are potent immunostimulants. In this study, CPG 7909 was formulated with the recombinant Plasmodium falciparum protein AMA1-C1 adsorbed to Alhydrogel (aluminum hydroxide) and used to immunize mice. Mice receiving free CPG 7909 in a separate same site injection to the AMA1-C1/Alhydrogel had the same antibody responses as mice receiving AMA1-C1/Alhydrogel alone. For mice immunized with CPG 7909 bound to the AMA1-C1/Alhydrogel formulation, there was a bell shaped CPG 7909 dose-response curve with the highest antibody response co-incident with the concentration of CPG 7909 that saturated binding to the Alhydrogel. At a higher CPG 7909 dose where 74% was unbound, there was no enhancement of response over AMA1-C1/Alhydrogel alone. Our results suggest that the adjuvant effects of CpGs are optimal when adsorbed to Alhydrogel and highlight the need for careful characterization of the vaccine formulation.

Fig. 1

Antibody responses in mice to AMA1-C1/Alhydrogel vaccines containing 30 ng AMA1-C1 and various amounts of unbound (A) or bound (B) CPG 7909 per dose. The formulation containing 50 μg of partially bound CpG (B) consisted of 26% and 35% bound CpG for the day 0 and 28 immunizations, respectively (determined by spectrophotometry). Arithmetic mean antibody in the groups of 9-10 mice were determined (bars). Individual mice are indicated as small circles.

Fig. 2

IgG subclass determination in sera from selected groups of mice. AMA1-C1/Alhydrogel vaccines contained various amounts of unbound, partially bound or bound CPG 7909. Anti-AMA1-FVO-specific IgG1, IgG2a, IgG2b, and IgG3 levels were measured by ELISA using normalized sera and secondary antibody conjugates. Bars represent the arithmetic means of optical density readings of day 43 sera from groups of 9-10 mice with values for individual mice indicated with small circles.